genotoxic effect of methotrexate on bone marrow ...egyptianjournal.xyz/64_10.pdfmouse mus musculus....

The Egyptian Journal of Hospital Medicine (Jul. 2016) Vol. 64, Page 350- 363

350

Received: 02/07/2016 DOI : 10.12816/0029027

Accepted: 12/07/2016

Genotoxic Effect of Methotrexate on Bone Marrow Chromosomes and DNA

of Male Albino Mice (Mus musculus) Nagla Zaky Ibrahim El –Alfy

1, Mahmoud Fathy Mahmoud

1,

Amany Ibrahim Alqosaibi2 and Sally Ramadan Gabr El-Ashry

1

1Biological and Geological Sciences Department, Faculty of Education, Ain Shams University, Cairo,

Egypt, 2Biology Department, Science College, University of Dammam, Dammam, Saudi Arabia

* Corresponding Author: Mahmoud Fathy Mahmoud, Biological and Geological Sciences Department,

Faculty of Education, Ain Shams University, Egypt [email protected]

ABSTRACT

Aim of the work-Methotrexate (MTX), a structural analogue of folic acid, is an antineoplastic and

antirheumatic agent which is used in a variety of clinical schedules and combination therapy regimens in

man. Material and methods- Sixty mice of nearly the same age were randomly categorized into four

groups (one control and three treated groups with different doses of methotrexate). Mice of the treated

groups 1, 2 and 3 were intraperitoneally injected with a single dose of methotrexate (2.5, 5 or 10 mg/kg b.

wt. respectively) at the first day of the experiment. All the control and the treated animals were sacrificed

after 24, 48 or 72 hour by cervical dislocation post treatment. Results-Methotrexate treatment induced

structural and numerical chromosomal aberrations in male mice bone marrow cells which were

significantly increased (P< 0.001) by dose and time. Structural aberrations were chromosomal gap,

fragment, break, centromeric attenuation, deletion, centric fusion, ring formation, end to end association

and beaded chromosomes. Numerical aberration was polyploidy. Also, methotrexate treatment decreased

the mitotic index in bone marrow cells of all the treated mice in comparison with the control group by

increasing dose and time of treatment. Comet assay results indicated that treatment with methotrexate

significantly increased (P< 0.001) DNA damage in the blood leukocytes in dose and time dependent

manner. Conclusion- It can be concluded that methotrexate induced genetic damage on the chromosomes

and DNA content of male albino mice even after single treatment with low doses .

Keywords: Methotrexate, Mice, Chromosomes, DNA, Comet assay.

INTRODUCTION Cancers are a group of diseases

characterized by uncontrolled cell growth and

spread [1]

. Methotrexate (MTX), is one of the

widely used antineoplastic drug and a well

known immunosuppressant introduced for

therapeutic use since 1950s [2]

. It is used against

a broad range of neoplastic disorders including

acute lymphoblastic leukaemia, non-Hodgkin’s

lymphoma, breast cancer and testicular tumors [3]

. Further, it is effective for the treatment of

psoriasis, rheumatoid arthritis and different

immune suppressive conditions [4]

. It is also one

of the drugs of choice in the new regimen

combination treatment against rheumatoid

arthritis and for several tumors [5]

. It was proved

that high dose of MTX regimens can be used

against primary central nervous system

lymphomas as well as liver cholestatic disorders [6]

. The basic principle of therapeutic efficacy of

MTX is due to the inhibition of dihydrofolate

reductase, a key enzyme in the folic acid

metabolism, which converts dihydrofolic acid to

tetrahydrofolic acid. The perturbation in the

folic acid metabolism leads to depletion of

nucleotide precursors like thymidylates and

purines, which in turn inhibits DNA, RNA and

protein synthesis. MTX also inhibits

thymidylate synthase and the transport of

reduced folates into the cell [7]

. MTX was found

to be a clastogenic agent in tumor cells and in

cultured mammalian cells [8, 9]

. The

carcinogenicity, mutagenicity, teratogenicity and

embryo lethality of MTX in different test

systems have been studied and reviewed and the

data have been listed in the genetic activity

mailto:[email protected]

Genotoxic Effect of Methotrexate on Bone Marrow Chromosomes…

351

profile (GAP) database [10]

. Mardini and

Record [11]

reported that the most serious side

effect of MTX therapy is hepatic toxicity.

Dadhania et al. [12]

reported that MTX increased

the intestinal toxicity in rat that assessed by

evaluating different parameters of oxidative

stress and DNA damage. Padmanabhan et al.

[13] investigated that MTX decreased the sperm

count and increased the frequency of sperms

with abnormal head. Del Campo et al. [14]

proved that MTX is also a potent teratogen.

Beluret al. [15]

proved the hematologic and

myelo -suppressive effects of MTX. Pellizzer et

al. [16]

examined the developmentally toxic

effects of MTX in a variety of other vertebrate

model organisms such as mice, rats and rabbits.

Kasahara et al. [17]

reported that MTX is

considered as a weak clastogen as it induced

chromosomal aberrations in bone marrow of

mouse only after multiple treatments. The aim

of this study is to investigate the genotoxic

effect of methotrexate on bone marrow

chromosomes and DNA content of male albino

mice.

MATERIAL AND METHODS

Animals:

Sixty mature male mice (Mus musculus)

of nearly the same age (16-18 weeks old) with

an average body weight (24 ± 2 g), obtained

from the closed colony of Theodor Bilharz

Research Institute, Cairo, were individually

weighed and randomly categorized into four

groups so that there were no statistically

significant differences among group body

weight means. Each group consisted of fifteen

mice. Mice were apparently normal and healthy.

They kept in animal house under suitable

conditions during the whole period of the

experiment. Animals were fed on standard

rodent pellet diet and supplied with water. These

animals were divided into four groups. One

group served as the control group (injected

intraperitoneally with 1 ml/kg distilled water)

and the other three groups (group1, group2 and

group3) served as the treated groups. Mice of

group1, group2 and group3 were injected

intraperitoneally with three different doses of

methotrexate drug (2.5, 5 or 10 mg/kg b.wt.

respectively, once on the first day of the

experiment). All the control and the treated

animals were sacrificed by cervical dislocation

after 24, 48 and 72 hr of treatment for collection

of samples.

Chemicals:

The drug used in the present investigation

was methotrexate (MTX) the active substance in

amethopterin .Its chemical structure is: L-

glutamic acid, N-(4-{2, 4-diamino-6-pteridinyl)

methyl)-nmethylamino} benzoyl with empirical

formula C20H22N8O5, in the form of solution for

injection (vial 50 mg MTX/ 2 ml distilled water)

produced by Orion Pharma, Orion Corporation,

Espoo, Finland. Appropriate methotrexate

solutions of the different concentrations were

prepared by dilution with distilled water, stored

at or below 25°C and protected from light. Its

chemical characteristics are represented by the

following structure [18]

as shown in figure 1.

Chromosomal Aberration Assay:

Bone marrow chromosome preparations

were carried out according to the method of

Preston et al.[19]

.

Mitotic index (MI):

It was calculated by counting the number

of dividing cells among at least 1000 cells

prepared for metaphase spreads of each group

(five animals per each group) and expressed in

percentage.

Comet assay :

Immediately after decapitation of animals,

1ml of blood was collected in an Eppendorf tube

containing 100μl of 10% EDTA. The blood

samples were stored in a dark box at 4ºC until

use. Peripheral blood leukocytes were isolated

by centrifugation (30 min at 1300xg) in Ficoll-

Paque density gradient (Pharmacia LKB

Biotechnology, Piscataway, NJ, USA). After

centrifugation, the leukocytes were aspirated

and washed twice by phosphate-buffered saline

at pH 7.4 (PBS). The comet assay was

performed according to the method of Singh et

al. [20]

with modifications according to Blasiak

et al. [21]

.

Statistical analysis:

The statistical software package, SPSS

version 16.0 for windows was used for all

statistical analysis. The study of chromosomal

aberrations and data of comet assay were

analyzed and expressed as mean and the

Nagla El –Alfy et al

352

standard deviation (mean ± SD). Each treated

group was compared to the control group with

independent samples t- test. Histograms of

cytogenetic data were drawn using Excel 2010.

The result was considered to be significant when

P is less than or equal to 0.05 or highly

significant when P is less than or equal to 0.001.

RESULTS

Chromosomal Aberration Assay:

The diploid number of the control male

mouse Mus musculus is 2n=38+XY. The

chromosomes of the mouse are telocentric and

their lengths are forming a continuous series. By

using classical techniques the majority of the

mouse chromosomes could be identified easily

in figure 2. The chromosomes are subdivided

into five distinct groups according to their

lengths. Each group is made up of chromosomes

having similar lengths figure 3. In the present

study, 500 well spread metaphases were

examined in each group to show the effects of

the three doses of methotrexate drug at 24, 48

and 72 hour post treatment on the bone marrow

cells respectively. There were two types of

chromosomal aberrations; the first one is

structural aberrations and the second is

numerical aberrations. The current results

indicated that the three doses of methotrexate

(2.5, 5 and 10 mg/kg b.wt.) induced both

structural and numerical aberrations in the

chromosomes of the bone marrow cells of the

mouse Mus musculus. The incidence of

structural and numerical chromosomal

aberrations were significantly increased (P<

0.001) over the control group table 2, figure 17.

The results clearly indicated that there

was a gradual significant increase in the mean of

various types of chromosomal aberrations with

increasing the dose and time intervals table 1

and figure 16. Structural chromosomal

aberrations that were induced in the

chromosomes of the bone marrow cells of Mus

musculus by the treatment with three different

doses of methotrexate were centromeric

attenuation (Ca) figures 5, 6, 10, 13 and 14,

centric fusion (Cf) figure 4, ring form

chromosome (R) figures 6, 8 and 11 and end to

end association (Ee) figures 5, 6, 11 and 13,

chromosomal gaps (Chg) figures 6 and 12,

chromatid gap (Cg) figures 8, 9, 11 and 14,

beaded chromosomes (Bch) figure 7 chromatid

deletion (D) figures 4, 6, 8, 9, 11, 12, 13 and 14

and chromatid fragmentation (F) figures 6, 8, 9,

11, 12 and 13. Numerical aberration that was

induced after the treatment with the tested doses

of methotrexate is in the form of polyploidy

figure 15. The mean of chromosome and

chromatid gaps, fragment, deletion and

centromeric attenuation were higher as

compared to other chromosomal aberrations as

shown in figure 17 and table 2.

At 24 hrs administration of methotrexate,

the total mean of chromosomal aberrations in

the treated mice were increased to 21.2±5.13,

31±4.13 and 50.4±5.7 when compared to

controls 10.8±3.53 respectively after

administration of 2.5, 5, 10 mg/kg b.wt. of

methotrexate. At 48 hrs of administration the

total mean of chromosomal aberrations in the

treated mice were 27±6.16, 46.2±6.89 and

67.4±6.7 respectively when compared to the

control which reached 10.8±3.53 for the various

doses of methotrexate with 2.5, 5, 10 mg/kg

b.wt. of methotrexate. At 72 hrs administration

of methotrexate the total mean of chromosomal

aberrations in the treated mice increased to

44.2±6.13, 70±5.06, 98.8±8.52 respectively

when compared to the control which reached

10.8±3.53 after the administration of 2.5, 5 and

10 mg/kg b.wt. of methotrexate .

The mitotic index:

In the present study, the mitotic index was

calculated and expressed in percentage to

evaluate the effect of methotrexate treatment on

cellular proliferation, as shown in table 4 and

figure 19 methotrexate administration resulted in

a reduction in the percent of dividing cells. The

reduction was proportional to both the dose and

the period after adminstration

Comet Assay:

In the present study the extent and

distribution of DNA damage in the blood

leukocytes was assessed using the comet assay

by counting the damaged cells out of 100 cells

on the slides for each animal. The percentages of

DNA damage were illustrated in table 3 as mean

± SD and graphically showed in figure 18. Table

3 showed a highly significant increase (P<

0.001) in the mean ± SD of DNA damage


353

percentages in all the three treated groups after

72 hr of MTX treatment and also in group 3 of

mice treated with 10 mg/kg b.wt. MTX after 24

and 48 hr of treatment as compared to the

control group .

The mean ± SD of DNA damage in the

treated mice was highly significant (P< 0.001)

and increased to 45±7.07, 62.5±3.54, and

72.5±3.54 respectively after 72 hr of treatment

with 2.5, 5 and 10 mg/kg MTX b.wt. as

compared to 4±1.41 in the control group. Images

of single cell gel electrophoresis were classified

according to the degree of damage after

migration through electrophoresis and visualized

by the digital camera fitted fluorescent

microscope as shown in figures. It revealed an

intact DNA in the control group figure 20; while

a high degree of DNA damage clarified by a

slightly pointed end due to the migration of

fragmented DNA through electrophoresis

(tailed) was presented in figures 21, 22 and 23.

Figure 18 showed that there were marked

variations in the mean of DNA damage in all the

three methotrexate treated groups as compared

to the corresponding control values. Also, it

indicated that there was a gradual increase in the

DNA damage with increasing the dose and time.

DISCUSSION

The present results of chromosomal

aberration assay showed that the incidence of

aberrant metaphases with chromosomal and

chromatid aberrations were significantly

increased (P< 0.001) in mice bone marrow cells

after 24, 48 and 72 hour of treatment with the

three treated doses of methotrexate 2.5, 5 and 10

mg/kg b.wt. respectively. These results are in

agreement with the reports of Alam et al.[22]

that

methotrexate treatment increased the mean of

chromosomal aberrations and percentage of

aberrant metaphases in mouse bone marrow.

Choudhury et al.[23]

recorded that the number

of aberrations are not linear with the increase in

the doses of methtrexate, in spite of that the

present study showed that the increase of the

mean of chromosomal aberrations in bone

marrow cells of treated animals over that of the

control animals was dose and time dependent

and statistically highly significant (P< 0.001).

The results of the current study showed that

methotrexate treatment induced certain types of

structural aberrations which were chromosomal

gap, fragment, break, centromeric attenuation,

deletion, centric fusion, ring formation, end to

end association and beaded chromosomes. Also,

MTX treatment caused numerical aberration in

the form of polyploidy. This result agreed with

study of Branda et al.[24]

;they reported that

methotrexate caused deficiency of folates by

interfering in the folate metabolism. The

deficiency of folates may lead to demethylation

of heterochromatin causing structural

centromere defects that could induce abnormal

distribution of replicated chromosomes during

nuclear division such as centromeric attenuation,

deletion, centric fusion, chromosomal gap, ring

formation, fragment and segmented

chromosomes. Because aneuploidy of

chromosomes 17 and 21 is often observed in

breast cancer and leukaemia and increased risk

for these cancers was associated with folate

deficiency [25]

. Sutherland [26]

observed that

folate deficient medium or methotrexate

treatment is standard methods of eliciting the

expression of fragile chromosomes sites in

human cells which have been implicated as

sources of chromosome aberrations. Also,

current results indicated that the mean of

chromosome and chromatid gaps, fragment,

deletion and centromeric attenuation were

higher as compared to other chromosomal

aberrations. This result can be explained

according to Stampfer et al.[27]

who confirmed

that folic acid deficiency caused fragments in

mice chromosomes and enhances tumor

development. Also, Henning et al.[28]

demonstrated that the deficiency of folate and

niacine produced fragments in cultured human

lymphocytes which would enhance the

development of liver cancer in rats fed a diet

deficient in methionine and choline. Alonso and

varela [29]

demonstrated that low folate intake or

high alcohol consumption due to deletion and

may negate some of the protective effects.

Nguyen et al. [30]

postulated the relationship

between the folate deficiency and increasing of

the percentage of fragments. Kasahara et al. [17]

reported that methotrexate is considered as weak

clastogen as it induced chromosomal aberrations

in bone marrow of mouse only after multiple


354

treatments. It was reportedly clastogenic in

human bone marrow (in patients) [31]

but was

nonclastogenic in human lymphocytes in vivo [32]

. Donya and Aly [18]

indicated that it is a

potent inducer of chromosomal aberrations in

mouse somatic and germ cells, MTX are found

statistically highly significant (P< 0.001) after

the treatment with the doses of 5 and 10 mg/kg

b.wt. of methotrexate, in single treatment and

were enhanced by multiple treatments.

However, the present study showed that

methotrexate induced chromosomal aberrations

per 500 metaphases in bone marrow cells of

mice after a single treatment with the three

doses (2.5, 5 and 10 mg/ kg) and at 24 hr, 48 hr

and 72 hr post-treatment. These induced

aberrations were statistically significant (P<

0.001) and highly significant (P< 0.05). This is

in agreement with the previous investigation of

Choudhury et al. [33]

who found that the

induced chromosomal aberrations (excluding

gaps) per hundred metaphases recorded from

bone marrow cells of mice at 24 hr post-

treatment with the three different doses (2, 10

and 20 mg/kg) of MTX were statistically highly

significant (P< 0.001). Thus, this indicates that

methotrexate was highly clastogenic in mouse

bone marrow even after a low dose single

treatment. The results of the present study

showed that the maximum chromosome

aberrations were recorded at 72 hr post-

treatment with 10 mg MTX/kg b.wt. This is in

agreement with the result of Alam et al. [22]

;

they observed that methotrexate treatment

caused a significant increase in chromosomal

abnormalities and the frequency of aberrant cells

and they observed also that the types of

aberrations were structural chromosomal

aberrations including chromatid breaks,

fragments, gaps and deletions besides

centromiric attenuation, endomitosis, centric

fusion and end to end association. Also,

numerical chromosome aberrations were

observed in the form of hypoploidy,

hyperploidy and polyploidy.

In the present study, we examined also

effect of methotrexate on DNA in the peripheral

blood leukocytes by using comet assay as it is a

sensitive, simple method to detect very low

levels of DNA damage [34]

. The Comet assay

was generally very sensitive in assessing

genotoxic damage, making it a good biomarker

of induced DNA damage [35]

. Therefore, it has

been widely used in studies on DNA repair,

genetic toxicology, radiation, pollution and

ageing [36]

. Martinez et al. [37]

suggested that the

modified comet assay proved to be a convenient

and sensitive biomonitoring tool for individuals

occupationally or voluntary exposed to tinner

inhalation. The data obtained from the present

study clearly showed a highly significant

increase (P< 0.001) in the mean ± SD of DNA

damage in all the three treated groups at 72 hr of

administration of methotrexate and also in group

3 of mice treated with 10 mg/kg methotrexate at

24 and 48 hr administration as compared to the

controls. We demonstrated that the effect of

methotrexate on DNA content was dose and

time dependent. This result can be explained by

the study of Genestier et al.[38]

.They reported

that the deficiency of folates caused by

methotrexate treatment was often associated

with genotoxic damage like strand breaks,

chromosomal abnormalities, defective DNA

repair, increased somatic mutation rates ,

inhibition of cell proliferation, disruption of cell

cycle and induction of apoptosis in susceptible

cells. Also, Hassab El-Nabi and Elhassaneen [39]

indicated that methotrexate induced

apoptosis (DNA fragmentation) in liver, spleen

and thymus of rat after 24 hr of treatment with

90 and 150 mg MTX/ kg b.wt. and the intensity

of DNA fragmentation increased with dose

dependent manner. A number of investigators

have examined the genotoxic effect of

methotrexate on DNA content by using comet

assay. Kopjar and Garaj-Vrhovac [40]

used the

alkaline comet assay to evaluate the

genotoxicity towards peripheral lymphocytes of

medical personnel regularly handling various

antineoplastic drugs, including

cyclophosphamide, vincristine, vinblastine, cis-

platinum, 5-fluorouracil, bleomycin, MTX and

adriamycin. Comet assay results of the study of

Kushwaha et al. [41]

indicated also significant

DNA damage (P< 0.05) in different organs as

liver, lung, heart, brain, kidney and bone

marrow cells induced by MTX as compared to

the control group after 28 days repeated oral

doses treatment (0.5, 1 and 2 mg/kg).


355

Padmanabhan et al. [42]

assessed DNA damage

and the cytotoxicity of methotrexate in the germ

cells using the sperm comet, halo and TUNEL

assay in testis. The sperm comet assay in the

study of Padmanabhan et al. [42]

revealed the

genotoxicity of MTX as observed by an increase

in the main parameters of the comet DNA

damage analysis.

According to this study, we can conclude

that methotrexate is highly clastogenic,

mutagenic and cytotoxic drug. It had very

harmful effect on chromosomes and DNA of

male Mus musculuseven after low doses and

single treatment.

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357

Table 1- The relationship between the mean of total chromosomal aberrations of all treated groups with

methotrexate (Group 1, 2 and 3) after 24, 48 and 72 hour of treatment.

24 hrs 48 hrs 72 hrs

Control 10.8 10.8 10.8

Group 1 21.2 27 44.2

Group 2 31 46.2 70

Group 3 50.4 67.4 98.8

Table 2- The relationship between mean of total chromosomal aberrations of male albino mice in the

control group and each of the treated groups with methotrexate (groups 1, 2 and 3).

Groups Ca Chg&Cg Cf R Bch Ee D F Po Total

Control 1.8 2.6 0.4 1.8 0.2 0 1.6 2.4 0 10.8

Group 1 5.33 8.67 0.73 3.13 0.73 0.8 5.06 5.93 0.4 30.78

Group 2 8.13 15.27 2.27 4.67 1.8 1.27 6 8 1.67 49.08

Group 3 10.13 20.2 4 6.2 1.93 2.06 10.86 14.2 2.6 72.18

Table 3- The Mean and standard deviation of DNA damage as revealed by comet assay of peripheral

blood leukocytes of the control and treated male Albino mice Mus musculus.

Groups Dose (mg/kg) Time (hour) DNA damage (mean ± SD)

Control 4±1.41

Group 1 2.5

24 9±1.41

48 12.5±3.54

72 45±7.07**

Group 2 5

24 14±5.66

48 17.5±3.54

72 62.5±3.54**

Group 3 10

24 18.5±2.12**

48 22.5±3.54**

72 72.5±3.54**

*Significant. (P< 0.05)** Highly significant. (P< 0.001)

Table 4- Mitotic index (MI) in 1000 bone marrow cells for each experimental group and its percentage after

24, 48 and 72 hour of treatment with 2.5, 5 and 10 mg/kg b.wt. of methotrexate and the control group.

Groups Dose (mg/kg) Time/hour Score of divided cells

/ total cells (1000)

Percentages of

mitotic index

Control 180 18%

Group 1

2.5

24 hr 140 14%

48 hr 126 12.6%

72 hr 112 11.2%

Group 2

5

24 hr 104 10.4%

48 hr 92 9.2%

72 hr 88 8.8%

Group 3 10

24 hr 50 5%

48 hr 38 3.8%

72 hr 12 1.2%


358

Figur

e 1: The chemical structure of methotrexate

Figure 2- Photomicrograph of metaphase chromosomes of the control male albino mouse Mus musculus

showing the diploid chromosome number 2n=38+XY. All chromosomes are telocentric.

)X: 2500(

Figure 3- The karyotype of male albino mouse Mus musculus.

①

② ③


359

Figure 4- Photomicrograph of metaphase chromosomes of male albino mouse Mus musculus after 24

hours of treatment with methotrexate (2.5 mg/kg b.wt.) showing centric fusion (CF) and deletion (D).

(X: 2500)

Figure 5- Photomicrograph of metaphase chromosomes of male Albino mouse Mus musculus after 24

hours of treatment with methotrexate (5 mg/kg b.wt.) showing centromeric attenuation (Ca) and end to

end association (Ee).

(X: 2500)


hours of treatment with methotrexate (10 mg/kg b.wt.) showing ring chromosome (R), deletion (D),

centromeric attenuation (Ca), end to end association (Ee), chromosomal gap (Chg) and fragment (F).

(X: 2500)


hours of treatment with methotrexate (10 mg/kg b.wt.) showing beaded chromosomes (Bch).

(X: 2500)

④ ⑤

⑥ ⑦


360


hours of treatment with methotrexate (2.5 mg/kg b.wt.) showing fragment (F), chromatid gap (Cg),

deletion (D) and ring (R).

(X: 2500)


hours of treatment with methotrexate (5 mg/kg b.wt.) showing fragment (F), chromatid gap (Cg) and

deletion (D). (X: 2500)


hours of treatment with methotrexate (10 mg/kg b.wt.) showing centromeric attenuation (Ca), it looks like

polyploidy. (X: 2500)

Figure 11- Photomicrograph of metaphase chromosomes of male albino mouse Mus musculus treated

with methotrexate (10 mg/kg b.wt.) for 48 hours showing end to end association (Ee), fragment (F),

chromatid gap (Cg), deletion (D) and ring chromosome (R).

(X: 2500)

⑧ ⑨

⑩ ⑪


361


with methotrexate (2.5 mg/kg b.wt.) for 72 hours showing fragment (F), chromosomal gap (Chg) and

deletion (D). (X: 2500)


with methotrexate (5 mg/kg b.wt.) for 72 hours showing centromeric attenuation (Ca), fragment (F), end

to end association (Ee) and deletion (D).

(X: 2500)


hours of treatment with methotrexate (10 mg/kg b.wt.) showing chromatid gap (Cg), centromeric

attenuation (Ca) and deletion (D).

(X: 2500)


with methotrexate (10 mg/kg b.wt.) after 72 hours of treatment showing polyploidy (Po). (X: 2500)

⑫ ⑬

⑭ ⑮


362

Figure 16- Histogram represents the relationship between the mean of total chromosomal aberrations of

all treated groups with methotrexate (group 1, 2 and 3) after 24, 48 and 72 hour of treatment.

Figure 17- Histogram represents the relationship between the mean of chromosomal aberrations of male

albino mice in all the treated groups with methotrexate (group 1, 2 and 3) after 72 hours of treatment

Figure 18- Histogram represents the relationship between the mean ± SD of DNA damage in peripheral

blood leukocytes of male Albino mice by using comet assay in the control and all the treated groups with

methotrexate after 24, 48 and 72 hour of treatment.

⑱

⑲

⑰

⑯


363

Figure 19- Chart represents the percentages of mitotic indices (MI) in all methotrexate treated groups and

the control group after 24, 48 and 72 hour of treatment, as calculated in table 3.

Figure 20- Photomicrographs of comet assay of mouse lymphocytes in the control group showing no or

minimum DNA migration. (X: 1500)

Figure 21- Photomicrographs of comet assay of mouse lymphocytes of group 1 which was treated with

2.5 mg/kg b.wt. of methotrexate after 72 hr of treatment showing slight DNA migration (short tails) in the

lymphocytes of mice . (X: 1500)

Figure22- Photomicrographs of comet assay of mouse lymphocytes of group 2 which was treated with 5

mg/kg b.wt. of methotrexate after 72 hr of treatment showing extensive DNA migration (long tails).

(X: 1500)

Figure 23-Photomicrographs of comet assay of mouse lymphocytes of group 3 which was treated with 10

mg/kg b.wt. of methotrexate after 72 hr of treatment showing the most extensive DNA migration (very

long tails). (X: 1500)

The symbols “-“and“+” represent cathode and anode, respectively, during the electrophoresis.

⑳ V

W

genotoxic effect of methotrexate on bone marrow ...egyptianjournal.xyz/64_10.pdfmouse mus musculus....

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